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by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating

frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can

pro-tect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that

expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated

in silico

using 2,145 field-sourced

H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and

also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6

months) protection postvaccination. Both neutralizing antibodies and antigen-specific CD4

and CD8

T cells were still

de-tected at 5 months postvaccination, suggesting that MVA-H5M provides long-lasting immunity.

IMPORTANCE

Influenza viruses infect a billion people and cause up to 500,000 deaths every year. A major problem in combating influenza is

the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel

in silico

mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a

consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent

T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza virus H5

hem-agglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as induce broader

immu-nity than a wild-type hemagglutinin.

I

nfluenza viruses are significant health concerns for animals and

humans. The World Health Organization estimates that every year

influenza viruses infect up to 1 billion people, with 3 million to 5

million cases of severe disease and 300,000 to 500,000 deaths

occur-ring annually (

1

). Highly pathogenic avian influenza (HPAI) H5N1

viruses have spread as far as Eurasia and Africa since their first

emer-gence in 1996. These viruses infect a range of domestic and wild avian

species as well as mammals (

2

) and pose a pandemic threat (

3

).

Cur-rent prevention and treatment strategies for H5N1 virus either are

antiviral or vaccine based or involve nonpharmaceutical measures,

such as patient isolation or hand sanitation (

4

,

5

). However, these

approaches have flaws (

5–7

), such that a broadly effective strategy for

H5N1 control remains elusive.

A powerful tool for preventing future H5N1 pandemics would

be a universal H5N1 vaccine. The generation of inactivated

vac-cines (INVs) has been optimized for seasonal flu but presents

several challenges for H5N1 viruses, including the following: (i)

the continual evolution of the viruses makes predicting a vaccine

strain difficult, (ii) egg propagation of vaccine stock is hindered

due to the high lethality of H5N1 viruses to eggs and the poultry

that provide them, and (iii) the 6- to 9-month time period

re-quired to produce INVs may be too long to protect large

popula-tions during a pandemic. In addition, initial studies in mice and

ferrets and phase 1 human clinical trials have demonstrated that

INVs and other split-virion vaccines may require higher doses of

antigen than traditional INVs, with more than one administration

being needed to provide protective immunity (

8

,

9

). Live vaccines

elicit both humoral and cellular immune responses. However,

they are not recommended for use in infants or in elderly or

immu-nocompromised individuals because they can cause pathogenic

reac-tions (

10

,

11

). Moreover, the viruses in live vaccines can revert to

wild-type (wt) viruses, potentially leading to vaccine failure and

dis-ease outbreaks (

12

). Thus, the development of new vaccine vectors

and novel approaches to antigen expression are urgently needed to

generate an effective H5N1 vaccine with broad cross-protective

effi-cacy. The modified vaccinia virus Ankara (MVA) vector offers several

advantages, such as (i) safety, (ii) stability, (ii) rapid induction of

humoral and cellular responses, and (iv) the ability to be given by

multiple routes of inoculation (

13–15

). In addition, we and others

have previously demonstrated the suitability of using MVA as a

vac-cine vector against H5N1 viruses (

14

,

16

).

Multiple approaches to the development of a universal

influ-enza vaccine that could be applied to H5N1 viruses have been

studied. One approach is to use conserved sequences, such as the

stalk region of hemagglutinin (HA) (

17–19

) or the internal

nu-cleoprotein (NP) or M1 protein (

20

,

21

). Another approach

in-volves consensus sequences that combine many H5N1

hemagglu-Received29 May 2014Accepted26 August 2014

Published ahead of print10 September 2014

Editor:D. S. Lyles

Address correspondence to Jorge E. Osorio, osorio@svm.vetmed.wisc.edu.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01532-14

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tinin sequences into a single gene. Of these approaches, only the

approach with consensus sequences has been shown to provide

partial protection against a diverse panel of H5N1 isolates (

22

).

Recently, a novel

in silico

mosaic approach has been shown to

provide broad and robust protection against highly variable viruses

(

23

). The method uses a genetic algorithm to generate, select, and

recombine potential CD8

T cell epitopes into mosaic proteins that

can provide greater coverage of global viral variants than any single

wild-type protein. This approach has been able to achieve between

74% and 87% coverage of HIV-1 Gag sequences, whereas a single

natural Gag protein achieved only 37% to 67% coverage (

23–25

).

Results in studies with rhesus monkeys showed that mosaic

se-quences increased both the breadth and the depth of cellular immune

responses compared to the breadth and the depth of the responses

achieved with consensus and natural sequences (

23

).

In this study, we used an MVA vector to express a mosaic

H5N1 HA gene. In mice, a single dose of an MVA construct that

expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M)

provided sterilizing immunity (no virus was detectable in lung

tissues postchallenge) against H5N1 HPAI clade 0, 1, and 2

vi-ruses. Furthermore, MVA-H5M provided full protection at as

early as 10 days postexposure and for as long as 6 months

postvac-cination. Both neutralizing antibodies and antigen-specific CD4

and CD8

T cells were detected at 5 months postvaccination. In

addition, MVA-H5M also provided cross-subtype protection

against H1N1 virus (A/Puerto Rico/8/34 [PR8]) challenge. Our

results indicate that the mosaic vaccine approach has great

poten-tial for broadening the efficacy of influenza vaccines, perhaps

in-cluding protection against multiple influenza virus subtypes.

MATERIALS AND METHODS

Cells and viruses.Chicken embryo fibroblasts (CEFs) and Madin-Darby canine kidney (MDCK) cells were obtained from Charles River

Laborato-ries, Inc. (Wilmington, WA, USA), and the American Type Culture Col-lection (ATCC; Manassas, VA, USA), respectively. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. CEFs were used for propagating MVA. Highly pathogenic avian influenza (HPAI) H5N1 virus A/Vietnam/ 1203/04 (A/VN/1203/04) was kindly provided by Yoshihiro Kawaoka (University of Wisconsin—Madison, Madison, WI, USA). Highly patho-genic avian influenza H5N1 viruses A/Hong Kong/483/97 (A/HK/483/ 97), A/Mongolia/Whooper swan/244/05 (A/MG/244/05), and A/Egypt/ 1/08 and seasonal influenza viruses, including A/Puerto Rico/8/34 (PR8; H1N1) and A/Aichi/2/1968 (H3N2), were kindly provided by Stacey Schultz-Cherry and Ghazi Kayali (St. Jude Children’s Research Hospital, Memphis, TN, USA). All viruses were propagated and titrated in MDCK cells with DMEM that contained 1% bovine serum albumin and 20 mM HEPES. Viruses were stored at⫺80°C until use. Viral titers were deter-mined and expressed as 50% tissue culture infective doses (TCID50s). All experimental studies with HPAI H5N1 viruses were conducted in a bio-safety level 3⫹(BSL3⫹) facility in compliance with the University of Wisconsin—Madison Office of Biological Safety.

Plasmid and MVA recombinant vaccine construction.A total of 3,069 HA protein sequences from H5N1 viruses available in the National Center for Biotechnology Information (NCBI) database were downloaded and screened to exclude incomplete and redundant sequences. The resulting 2,145 HA sequences were selected to generate one mosaic protein sequence, as previously described (26). Briefly, all 2,145 HA sequences were uploaded into the Mosaic Vaccine Designer tool webpage (http://www.hiv.lanl.gov/content /sequence/MOSAIC/makeVaccine.html). In the parameter options, the cocktail size was set to 1 in order to generate a single peptide that repre-sented all uploaded sequences. The rare threshold was set to 3 for optimal value, and most importantly, the epitope length parameter was set to an amino acid length of 12-mer in an attempt to match the length of natural T helper cell epitopes (27). The resulting mosaic H5N1 sequence (H5M) was back-translated and codon optimized for mice. The optimized H5M sequence was then synthesized commercially (GenScript USA Inc., Pisca-taway, NJ, USA). Transfer plasmid PI2-Red encodingDiscosomasp. red

FIG 1The mosaic H5 hemagglutinin (H5M) sequence. The mosaic H5N1 hemagglutinin (H5M) sequence was deduced from 2,145 HA sequences. The lines

above the sequence indicate known T helper cell, B cell, or cytotoxic T lymphocyte (CTL) epitopes that were found in H5M by use of the Influenza Research Database (IRD).

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(Madison, WI, USA).

Analysis of HA expressed by H5M.The hemagglutinin expressed by MVA-H5M was analyzed by Western blot analysis. CEF cells were infected at a multiplicity of infection (MOI) of 1 PFU/cell of the MVA-H5M and MVA-HA constructs and a construct consisting of MVA expressing lucif-erase (MVA-LUC). Infected cell pellets were harvested at 48 h postinfec-tion and lysed with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Protein was fractionated via SDS-PAGE and transferred onto a nitrocellulose membrane for hemagglutinin detection by a specific an-ti-HA antibody (Ab). 3,3=,5,5=-Tetramethylbenzidine (TMB) was used to visualize the HA protein in the membranes.

Functional analysis of H5M was done by hemagglutination assay (29). CEF cells were infected at an MOI of 1 with MVA-H5M, MVA-HA, and MVA-LUC. At 48 h postinfection, cells were harvested and 2-fold dilu-tions with phosphate-buffered saline (PBS) were made in round-bottom 96-well plates. Chicken red blood cells (RBCs) were added into each well, and the plates were incubated for 30 min. Lattice formations, which are indicative of the ability of HA to agglutinate RBCs, were observed in positive wells.

Animal studies.All mouse studies were conducted at the University of Wisconsin—Madison animal facilities and were approved by the Interin-stitutional Animal Care and Use Committee (IACUC). Challenge exper-iments involving H5N1 viruses were conducted at animal BSL3⫹ (ABSL3⫹) facilities. Challenge studies with seasonal influenza, PR8 (H1N1), and A/Aichi/2/1968 (H3N2) viruses were conducted under BSL2 conditions to facilitate animal monitoring.

Vaccine efficacies.In the first study, groups of 5-week-old BALB/c mice were vaccinated with 1⫻107PFU of either recombinant MVA-H5M or MVA-LUC via the intradermal (i.d.) route (8 mice per group). Intra-dermal inoculations were done by injecting 50␮l of PBS containing virus into footpads. At 4 weeks after vaccination, blood samples were collected for serological analysis. At 5 weeks postvaccination, mice were challenged by intranasal (i.n.) instillation, while they were under isoflurane anesthe-sia, with 100 50% lethal doses (LD50s) of A/Vietnam/1203/04 (1⫻104 TCID50s), A/Hong Kong/483/97 (4 ⫻ 103 TCID

50s), A/Mongolia/ Whooper swan/244/05 (1⫻103TCID

50s), or A/Egypt/1/08 (3.56⫻104 TCID50s) contained in 20␮l of PBS. Two mice from each group were euthanized at day 5 postchallenge, and lung tissues were collected for viral titrations and histopathology. For virus isolation, lung tissues were minced in PBS using a mechanical homogenizer (MP Biochemicals, So-lon, OH, USA), and the viral titers in the homogenates were quantified by plaque assay on MDCK cells. The remaining lung tissue was fixed in 10% buffered formalin. The remaining animals in each group (6 mice per group) were observed daily for 14 days, and survival and clinical param-eters, including clinical score and body weight, were recorded. Mice show-ing at least a 20% body weight loss were humanely euthanized.

The second study evaluated the protective efficacies of the MVA-H5M vaccine against seasonal influenza virus, PR8 (H1N1), or A/Aichi/2/1968 (H3N2). Groups of 5-week-old BALB/c mice were vaccinated with MVA-H5M or MVA-LUC as described above (8 mice per group). At 4 weeks after vaccination, blood samples were collected for serological analysis. At 5 weeks postvaccination, the mice were challenged, while they were under isoflurane anesthesia, by i.n. instillation of 50␮l of PBS containing 100 LD50s of PR8 (6.15⫻103TCID50s) or A/Aichi/2/1968 (3.405⫻106 TCID50s). Two mice from each group were euthanized at day 3 postchal-lenge, and lung tissues were collected as described above. The remaining

animals in each group (6 mice per group) were observed daily for 14 days as described above.

In the third study, we evaluated the ability of the MVA-H5M vaccine to confer both short- and long-term protection against avian influenza virus A/Hong Kong/483/97; this strain showed the most virulence from the previous study. Groups of 5-week-old BALB/c mice were vaccinated with MVA-H5M or MVA-LUC as described above (7 mice per group). Blood samples were collected at 10 days and 6 months postvaccination for short- and long-term studies, respectively. At 10 days and 6 months post-vaccination, mice were challenged, while they were under isoflurane an-esthesia, by i.n. instillation of 100 LD50s of A/Hong Kong/483/97 (4⫻103 TCID50s). Two mice from each group were euthanized at day 5 postchal-lenge, and lung tissues were collected for viral titrations and histopathol-ogy. The remaining animals in each group (5 mice per group) were ob-served daily for 14 days, as described above.

FIG 2The MVA-H5M vaccine expresses a higher level of protein than MVA

expressing wild-type hemagglutinin (MVA-HA) and elicits broad neutralizing antibodies against avian influenza viruses. (A) Western blot analysis of CEF cell lysates infected with MVA-H5M, MVA-HA, and MVA-LUC (negative control). HA from MVA-H5M was expressed as a cleavable protein that was the same as the HA from MVA-HA. The sizes of HA clade 0 (HA0), HA clade 1 (HA1), and HA clade 2 (HA2) are 75 kDa, 50 kDa, and 25 kDa, respectively. (B) The titers of neutralizing antibodies against influenza virus A/VN/1203/04, A/MG/244/05, A/HK/483/97, A/Egypt/1/08 (EGYPT/01/08), PR8 (H1N1), or A/Aichi/2/1968 (H3N2) virus in vaccinated mice (n8 mice per group) were measured at 4 weeks postvaccination. No statistically significant differences between titers against avian influenza viruses (P0.05, one-way ANOVA) were found.

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Serology.Serum antibody titers were determined by microneutraliza-tion assay. Briefly, serum was incubated at 56°C for 30 min to inactivate complement and then serially diluted 2-fold in microtiter plates. Two hundred TCID50s of virus were added to each well, and the plates were incubated at 37°C for 1 h. The virus-serum mixture was added to dupli-cate wells of MDCK cells in 96-well plates, the plates were incubated at 37°C for 72 h, and then the cells were fixed and stained with 10% (wt/vol) crystal violet in 10% (vol/vol) formalin to determine the TCID50. The titer was determined as the serum dilution resulting in the complete neutral-ization of the virus.

Histopathology and immunohistochemistry.Lung tissue samples for histological analysis were processed by the histopathology laboratory at the School of Veterinary Medicine, University of Wisconsin—Madison (Madison, WI, USA), and stained with hematoxylin and eosin. For im-munohistochemistry, tissue sections were deparaffinized and rehydrated as previously described (30). Slides were treated with antigen retrieval buffer, followed up with treatment with 3% H2O2. The slides were placed in blocking solution and incubated in goat anti-HA (1/300 dilution; Bio-defense and Emerging Infections Research Resources Repository number NR-2705) avian influenza virus polyclonal antibody for 24 h. Secondary

horseradish peroxidase-conjugated antigoat antibody at a 1/5,000 dilu-tion was added onto the slides, and the slides were incubated for 1 h. Then, the slides were stained with 0.05% 3,3=-diaminobenzidine (DAB) sub-strate to visualize the presence of avian influenza virus antigens.

T cell responses.At 6 weeks and 5 months postvaccination, MVA-H5M-vaccinated mice were euthanized (3 mice per group) and spleens were aseptically removed. Splenocytes from individual animals were sus-pended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 100␮g/ml streptomycin, and 0.14 mM␤-mercaptoethanol. Red blood cells were lysed with 1⫻BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, USA). Following washing with RPMI 1640 medium, cells were resuspended in the same medium and 1⫻ 106splenocytes were surface stained with anti-mouse CD4 fluorescein isothiocyanate (RM4-5) and anti-mouse CD8a peridinin chlorophyll pro-tein (53-6.7) monoclonal antibodies (BD Biosciences, San Jose, CA, USA).

In order to study intracellular cytokine responses, 1⫻106splenocytes were plated onto a 96-well flat-bottom plate and stimulated with diverse H5N1 HA peptide pools (5␮g/ml) in a 200-␮l total volume for 16 h. Brefeldin A (BD GolgiPlug; BD Biosciences, San Jose, CA, USA) was

Egypt / 01/08

Egypt / 01 /08

Egypt / 01/08

FIG 3MVA-H5M provides broad protection against multiple clades of avian influenza virus. (A) Efficacies of a single dose of the MVA-H5M or MVA-LUC

vaccine against highly pathogenic avian influenza viruses: clade 0 influenza virus A/HK/483/97 virus, clade 1 influenza virus A/VN/1203/04, clade 2.2 influenza virus A/MG/244/05, and clade 2.2.1 influenza virus A/Egypt/1/08. Mice vaccinated with MVA-H5M showed 100% survival, and MVA-H5M prevented morbidity during the studies. (B and C) No viral titers were detectable in the lung at day 5 postinfection (B), and MVA-H5M-vaccinated mice maintained their weight throughout the studies (C). Vaccinated mice were challenged at 5 weeks postvaccination, 2 mice per group were sacrificed at day 5 postinfection, and survival data were monitored for 14 days (n⫽6 mice per group).

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added at a final concentration of 1␮g/ml for the last 5 h of incubation to block protein transport. Cells were stained intracellularly for gamma in-terferon (IFN-␥) allophycocyanin (XMG1.2) and interleukin-2 (IL-2) phycoerythrin (JES6-5H4) after surface staining for CD4 and CD8a. All antibodies were from BD Biosciences (San Jose, CA, USA), except where noted. The samples were acquired on a BD FACSCalibur flow cytometer and analyzed with FlowJo (v10.0.6) software (TreeStar Inc., Ashland, OR, USA). The background cytokine level from medium-treated groups was subtracted from the cytokine level for each treated sample. The frequency of cytokine-positive T cells was presented as the percentage of gated CD4⫹ or CD8⫹T cells.

Statistical analysis.Student’sttests and one-way analysis of variance (ANOVA) were used to evaluate viral lung titers, antibody titers, and T cell responses between groups. Survival analyses were performed to assess vaccine effectiveness against challenge viruses. Probability values of⬍0.05 were considered significant. GraphPad Prism (v6) software (La Jolla, CA, USA) was used for all statistical analyses.

RESULTS

Construction of pox-based H5N1 mosaic hemagglutinin

vac-cine.

A mosaic vaccine that targets the hemagglutinin protein of

influenza H5N1 virus was constructed (

Fig. 1

). The HA mosaic

was generated using an input of 2,145 HA sequences from H5N1

influenza viruses available in GenBank. To maximize T helper cell

epitope coverage, the

in silico

algorithm was set to an amino acid

length of a 12-mer (

27

). The mosaic H5 (H5M) sequence was

back-translated into DNA and cloned into modified vaccinia virus

Ankara (MVA) to generate MVA-H5M. Recombinant H5M was

expressed as cleavable HA that resembled wild-type (wt) HA from

avian influenza virus A/VN/1203/04, as shown in

Fig. 2A

.

Inter-estingly, the level of protein expression from MVA-H5M-infected

cell pellets was higher than that from MVA expressing wild-type

hemagglutinin from A/VN/1203/04 (MVA-HA) (

16

) (

Fig. 2A

).

Additionally,

in vitro

functional analysis of H5M resulted in

hem-agglutination of RBCs at levels similar to those for wt HA (A.

Kamlangdee, B. Kingstad-Bakke, and J. E. Osorio, unpublished

data). These data thus demonstrate the successful generation of an

MVA vector expressing a mosaic H5N1 HA gene.

Efficacy of MVA-H5M vaccine against influenza viruses.

To

determine whether antibodies to MVA-H5M had

virus-neutraliz-ing activity, mice were intradermally inoculated with MVA-H5M,

MVA-LUC, or PBS; and the titers of antibodies against A/VN/

1203/04, A/MG/244/05, A/HK/483/97, and A/Egypt/1/08

chal-lenge viruses in immunized mice were measured. At 4 weeks

postimmunization, MVA-H5M elicited significant neutralizing

antibody (Ab) titers and protected against all four H5N1 strains

after challenge (

Fig. 2B

and

3A

to

C

). The geometric mean titers

(GMTs) of Abs against A/VN/1203/04, A/MG/244/05, A/HK/483/

97, and A/Egypt/1/08 did not differ significantly (

n

8 per group,

P

0.05) (

Fig. 2B

). None of animals injected with the MVA-LUC

control survived the challenge (

Fig. 3A

to

C

). Notably, no virus

replication was detected in the lungs of any of the

MVA-H5M-vaccinated mice challenged with any of the four H5N1 viruses

(

Fig. 3B

). Furthermore, vaccination with MVA-H5M reduced the

lung pathology after challenge with avian influenza viruses (

Fig.

4A

to

D

). MVA-H5M-vaccinated mice showed no to mild lung

lesions, whereas the MVA-LUC-vaccinated groups did show lung

lesions (

Fig. 4E

to

H

). Lesions included thickening of the alveolar

wall, lung consolidation with white blood cell infiltration, necrosis

of alveolar walls, and pulmonary edema. Immunohistochemistry

staining revealed large quantities of viral antigen in the MVA-LUC

control group (

Fig. 5E

to

H

). In contrast, viral antigen was not

detected in the lungs of mice that received MVA-H5M (

Fig. 5A

to

D

). These data demonstrate the ability of MVA-H5M to confer

both broad and strong protection against multiple clades of avian

influenza viruses.

We also evaluated the ability of the MVA-H5M construct to

protect mice against seasonal influenza viruses. Vaccination

fol-lowed the same protocol used with H5N1 viruses, except that mice

were challenged with A/Puerto Rico/8/1934 (PR8; H1N1) and

A/Aichi/2/1968 (H3N2). At 4 weeks postvaccination, the titers of

neutralizing Abs against both seasonal influenza viruses (

n

8 per

group) were below detectable levels (

Fig. 2B

). Despite the lack of

detectable neutralizing Ab, MVA-H5M conferred complete

pro-tection against PR8 (H1N1), with no significant weight loss being

observed (

Fig. 6A

and

C

). In contrast, no protection against

A/Ai-chi/2/68 (H3N2) challenge was observed (

Fig. 6A

and

C

).

Regard-less of the strain tested, viral replication was observed in the lungs

of mice vaccinated with MVA-H5M and challenged with seasonal

influenza viruses; however, mice challenged with PR8 had

signif-icantly lower viral lung titers than mice vaccinated with the

MVA-LUC control (

Fig. 6B

). In contrast, vaccination with MVA-H5M

FIG 4MVA-H5M reduces lung pathology after challenge with avian influenza viruses. Tissue sections of the lungs of mice vaccinated with MVA-H5M (A to D)

or MVA-LUC (E to H) and challenged with A/VN/1203/04 (A and E), A/MG/244/05 (B and F), A/HK/483/97 (C and G), or A/Egypt/1/08 (D and H) virus are shown. MVA-H5M-vaccinated mice showed normal to mild lung lesions, whereas MVA-LUC-vaccinated mice showed severe lung lesions, including lung consolidation, leukocyte infiltration, thickening of the alveolar septa, and alveolar edema.

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had no effect on viral replication in the lungs of mice challenged

with A/Aichi/2/68 (

Fig. 6B

).

Short- and long-term immunity.

To assess the ability of the

MVA-H5M construct to confer both short- and long-term

immu-nities, groups of mice were vaccinated with a single dose of

MVA-H5M and then challenged at either 10 days or 6 months

postvac-cination. MVA-H5M provided full protection against a lethal dose

of A/HK/483/97 at both tested time points (

Fig. 7A

and

B

).

Neu-FIG 5MVA-H5M prevents viral replication in the lung after challenge with avian influenza viruses. Tissue sections of the lungs of mice vaccinated with

MVA-H5M (A to D) or MVA-LUC (E to H) and challenged with A/VN/1203/04 (A and E), A/MG/244/05 (B and F), A/HK/483/97 (C and G), or A/Egypt/1/08 (D and H) virus are shown. Lungs from mice were processed by immunohistochemistry with H5N1-specific antibody. Brown staining for viral antigen is indicated with arrowheads.

FIG 6MVA-H5M provides heterosubtypic protection against H1N1 virus. (A) Efficacies of a single dose of MVA-H5M or MVA-LUC vaccine against seasonal

H1N1 and H3N2 influenza viruses. MVA-H5M provided 100% protection with low weight loss against H1N1 virus but failed to prevent H3N2 virus infection. (B) MVA-H5M significantly lowered H1N1 viral replication in the lung compared to that in groups of mice that were challenged with H3N2 virus. (C) MVA-H5M prevented infection of vaccinated mice upon challenge with H1N1 virus, and the mice showed no to mild clinical signs and slight weight loss, but the vaccine failed to prevent infection upon challenge with H3N2 virus. Vaccinated mice were challenged at 5 weeks postvaccination, 2 mice per group were sacrificed at day 3 postinfection, and survival data were monitored for 14 days (n⫽6 mice per group).

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tralizing antibodies against A/HK/183/97 were also detected at

both 10 days and 6 months postvaccination (

Fig. 7C

).

Microneu-tralization assays and a challenge study were conducted using

A/HK/183/97 virus because it was the most virulent strain among

the four H5N1 strains used in the present study. Surprisingly, the

GMTs of Abs detected at 6 months postvaccination were

substan-tially higher than those detected at 4 weeks postvaccination (

Fig.

2B

and

7C

). In these animals, H5N1-specific IFN-

-releasing

CD4

and CD8

T cell responses were detected using flow

cytom-etry. IFN-

-releasing CD4

and CD8

T cells were found in

MVA-H5M-vaccinated mice 5 months after dosing (2 weeks

be-fore challenge in the long-term protection study), indicating a

long-term memory response (

Fig. 7D

).

Immune responses of mosaic and natural sequence vaccines.

We compared the immune responses to the mosaic vaccine

(MVA-H5M) to those to the wild-type or natural sequence

vac-cine (MVA-HA). Flow cytometry analyses of cytokine-producing

cells demonstrated that MVA-H5M-vaccinated mice had broader

and higher IFN-

-producing CD4

responses as well as broader

and higher IL-2-producing CD8

T cell responses than mice

vac-cinated with MVA-HA (

Fig. 8A

and

B

) after stimulation with

di-verse H5 HA peptide pools. Furthermore, the T cell responses

against H1N1 (PR8) peptides were analyzed. The results showed

that MVA-H5M elicited CD8

T cells responses to HA peptides

from H1N1 (PR8) virus, and the response was higher than that to

the MVA-HA wild-type vaccine (

Fig. 8C

). While MVA-H5M

elic-ited neutralizing antibodies against all viruses tested, MVA-HA

failed to elicit detectable responses against A/Egypt/1/08 virus

(

Fig. 8D

).

DISCUSSION

The rapid evolution of influenza viruses poses global health

chal-lenges necessitating the development of vaccines with broad

cross-protective immunity. Herein, we describe the development of a

broadly protective vaccine, MVA-H5M, based on a mosaic

epitope approach. The mosaic epitope approach minimizes

ge-netic differences between selected vaccine antigenic sequences and

circulating influenza virus strains, while it maximizes the overall

breadth of cross-protective immune responses. Our results

dem-onstrated that a single dose of MVA expressing a mosaic H5

hem-agglutinin (MVA-H5M) provided broad protection against

mul-tiple H5N1 viruses, including the highly pathogenic Egyptian

strains, and also an H1 subtype virus (PR8). The MVA-H5M

vac-cine provided robust and prolonged protection against a lethal

dose of a highly pathogenic avian influenza virus at as early as 10

days and as long as 6 months postvaccination.

In the past few years, commercially available vaccines have

failed to induce the expected level of protection against the

cur-rently circulating clade 2.2.1 in Egypt (

31

,

32

). It is very important

that an H5N1 influenza virus vaccine provide broad cross-clade

protection against these clade 2.2.1 viruses, particularly the

A/Egypt/1/08 strain, because this strain possess one of the four

mutations that are necessary to sustain human-to-human

trans-mission (

33

). The MVA-H5M vaccine provided complete

protec-tion against this H5N1 strain in mice. The ability to provide

com-plete protection against H5N1 viruses with a single dose is also

important for implementation; societal acceptance of a

single-dose vaccine would likely be higher than that of a multisingle-dose

vac-cine, especially during a pandemic.

On the basis of our data, there are several plausible hypotheses

to explain how our mosaic vaccine confers broad protection

against influenza virus challenge. One possible explanation is that

the 12-mer mosaic sequence captures more T helper cell epitopes,

in which case the broad protective ability of MVA-H5M likely

results from greater epitope coverage for the mosaic approach

than for previous approaches (

23

). This could translate to a higher

level of CD4

T cells and antibody responses broader than those

induced by the wild-type sequence (MVA-HA). This hypothesis is

supported by the fact that MVA-H5M showed broader IFN-

-FIG 7MVA-H5M provides short- and long-term immunities. (A and B) BALB/c mice were immunized with a single dose of MVA-H5M or MVA-LUC (n⫽7

mice per group). At 10 days or 6 months postvaccination, mice were challenged with a lethal dose of influenza virus A/HK/483/97. Survival data (A) and percent weight loss (B) were monitored for 14 days. (C) Neutralization titers from vaccinated mice at 10 days and 6 months postvaccination (n⫽7 mice per group). (D) CD4⫹and CD8⫹T cells responses at 5 months postvaccination.

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producing CD4

T cell epitope coverage and elicited broader

cross-clade neutralizing antibody responses (

Fig. 8A

,

B

, and

D

).

This MVA-H5M mosaic vaccine also elicited broader and higher

CD8

T cell responses than the wild-type (MVA-HA) vaccine

(

Fig. 8B

). However, other immunological aspects of the

MVA-H5M vaccine still need to be further characterized. For example,

cytokine profiles, antibody epitope coverage, and mapping would

all be necessary to fully understand the mechanism responsible for

protection. Also, challenge studies comparing the mosaic

(MVA-H5M) vaccine and the wild-type vaccine or a natural sequence

(MVA-HA) vaccine will be conducted in the future. However, the

in vitro

data from T cell and neutralizing antibody responses

strongly indicate the broader immunogenicity of the mosaic H5

than the wild-type H5; i.e., the mosaic H5 elicited broader epitope

responses for both CD4

and CD8

against H5N1 peptides (

Fig.

8A

and

B

). It also elicited higher CD8

T cell responses against

PR8 peptides (

Fig. 8C

) than wild-type H5 (MVA-HA).

Further-more, the mosaic H5 elicited broad neutralizing antibodies

against all 4 clades of avian influenza viruses that we tested,

in-cluding clade 0, clade 1, clade 2.2, and clade 2.2.1, whereas the

wild-type vaccine failed to elicit neutralizing antibodies against

clade 2.2.1 influenza virus A/Egypt/1/08, which is considered a

mutant strain (

31

,

32

,

34

). Commercially available vaccines also

failed to protect against this mutant Egyptian strain in Egypt in

recent years (

32

).

A second mechanism that may explain the breadth of

protec-tion conferred by the MVA-H5M vaccine is that the mosaic

ap-proach maintains an intact antigenic structure and, presumably,

physiological function (

35

). It has previously been reported that

most universal neutralizing antibodies are elicited by peptides in

the stalk regions (

36

,

37

). Our MVA-H5M vaccine has a normal

hemagglutination function and is also expressed as a cleavable

protein. Furthermore, our mosaic H5 might provide a higher level

of accessibility to the stalk region and stimulate a more robust

neutralizing antibody response against epitopes in the stalk

re-gion. Crystallography of the expressed mosaic H5 would likely be

required to reveal the actual structure of this protein and compare

it to the known structure of H5 hemagglutinin.

The MVA-H5M construct provided sterilizing lung protection

against H5N1 viruses, and no mortality or morbidity was detected

EGYPT /01 /08

FIG 8MVA-H5M elicits broader T cell epitope coverage, higher T cell responses, and broader neutralizing antibody responses than the wild-type

hemagglu-tinin-based vaccine. (A and B) Percentages of IFN-␥-producing CD4⫹T cells (A) and IL-2-producing CD8⫹T cells (B) from mice that were vaccinated with MVA-H5M or MVA-HA (n⫽3 mice per group). Splenocytes from vaccinated mice were collected and stimulated with HA peptide pools (indicated by p1 to p10) from H5N1 viruses. (C) IFN-␥-producing CD8⫹T cells from mice that were vaccinated with MVA-H5M or MVA-HA (n⫽3 mice per group). Splenocytes from vaccinated mice were collected and stimulated with HA peptide pools (indicated by p1 to p9) from H1N1 (PR8) virus. (D) Titers of neutralizing antibodies against H5N1 viruses elicited by the MVA-H5M and MVA-HA vaccines (n⫽7 mice per group). *,P⬍0.05, Student’sttest; **,P⬍0.01, Student’sttest.

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play a role.

It is currently unclear whether the use of a live viral vector such

as MVA contributed to the increased cross protection described

herein. It is possible that H5M expression by MVA induced high

levels of cross-reactive neutralizing antibodies as well as CD4

and CD8

T cells via innate immune activations (

39

). In future

studies, we will evaluate the protective efficacy of the H5M antigen

as a recombinant protein without the MVA vector. This approach

will likely require the use of adjuvants and multiple doses to

achieve the desired protection.

The mosaic approach has previously been used for developing

vaccines against the highly variable HIV strains, capturing

poten-tial CD8

T cell epitopes with a length of 9 amino acids (

23

,

26

)

while still maintaining a normal protein structure. Because

com-plete protection against influenza viruses is based primarily on

humoral immunity (

40

), we modified the algorithm for epitopes

of 12 amino acids in order to capture potential T helper cell

epitopes (

27

) in order to target antibody-producing plasma cell

via T helper cell activation. We speculate that this strategy may

have facilitated achievement of a high neutralizing antibody titer

with a single dose of MVA-H5M (

Fig. 2B

).

The vaccine elicited strong humoral responses against multiple

H5N1 viruses but no cross-neutralizing antibodies against

sea-sonal influenza viruses (H1N1 and H3N2). Despite the lack of

neutralizing antibodies against H1N1 PR8 virus, the MVA-H5M

vaccine provided 100% protection against the PR8 virus. This

sug-gests a substantial role of cellular immune responses against the

PR8 virus, as shown in

Fig. 8C

, likely because the H5M protein

possesses some cytotoxic T lymphocyte epitopes of the PR8 virus

(

35

). This protection can also be explained by the genetic

relation-ship between the H5 and H1 hemagglutinin subtypes, as both

belong to group 1 influenza A virus hemagglutinin (

41

), and it

elicits a large amount of nonneutralizing antibody which then

targets and destroys H1N1 virus via an antibody-dependent

cel-lular cytotoxicity (ADCC) mechanism (

42

). However, the

MVA-H5M vaccine did not protect immunized mice against influenza

virus A/Aichi/2/68, which is likely due to antigenic differences, as

H3 belongs to group 2 influenza A virus hemagglutinin. Because

the MVA vector can be designed to contain multiple inserts,

fu-ture constructs will contain mosaics from several hemagglutinin

subtypes, including important seasonal (e.g., H3) and emerging

(e.g., H7) pathogens. Since this vaccine provides broad protection

and a long duration of immunity, utilizing an MVA vector

ex-pressing seasonal mosaics might diminish the need for annual

vaccination.

The ability of the MVA-H5M vaccine to confer broad

protec-tive immunity against various heterologous strains as well as

het-erosubtypic strains makes the mosaic approach a very promising

strategy to combat the antigenic diversity of influenza viruses.

Taken together with codon optimization of HA for a high level of

protein expression and the use of an MVA vector as a backbone for

cellular immunity activation, this approach promises to increase

assistance with T cell analyses and Matthew Aliota (Pathobiological Sci-ences, University of Wisconsin—Madison, Madison, WI, USA) for criti-cal review of the manuscript.

A.K., B.K.-B., and J.E.O. conceived and designed the experiments. A.K. and B.K.-B. performed the experiments. A.K. analyzed the data. T.K.A., T.L.G., and J.E.O. contributed reagents, materials, and analysis tools. A.K., B.K.-B., and J.E.O. wrote the paper.

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Figure

FIG 1 The mosaic H5 hemagglutinin (H5M) sequence. The mosaic H5N1 hemagglutinin (H5M) sequence was deduced from 2,145 HA sequences
FIG 2 The MVA-H5M vaccine expresses a higher level of protein than MVAexpressing wild-type hemagglutinin (MVA-HA) and elicits broad neutralizingantibodies against avian influenza viruses
FIG 3 MVA-H5M provides broad protection against multiple clades of avian influenza virus
FIG 4 MVA-H5M reduces lung pathology after challenge with avian influenza viruses. Tissue sections of the lungs of mice vaccinated with MVA-H5M (A to D)or MVA-LUC (E to H) and challenged with A/VN/1203/04 (A and E), A/MG/244/05 (B and F), A/HK/483/97 (C and
+4

References

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Due to its electron configuration (Xe) 4 f 14 6 s 2 , Ytterbium (Yb) could form divalent oxide, YbO. In this study, the solid-state synthesis of metastable YbO was investigated

Multilateral funding agencies (e.g. the Global fund to Fight AIDS, TB and Malaria) and bilateral funding agencies (e.g. USAID) committed to TB control should provide investment

.Binding of iron to the insoluble fraction of albumen in the absence of acids • or in the presence of citric acid CJ and ascorbic acid• at different pH values... Binding of iron to

Purpose: The current project evaluated the impact of a short-term, supported funding initiative that allowed staff from allied health (AH) professions to undertake research